Erythritol is a non-cariogenic sweetener. Currently, it is primarily produced through microbial fermentation using glucose as the main substrate. However, this method suffers from limitations, such as low yield and conversion rate. Glycerol, being an abundant and low-value renewable resource, will be utilized as substrate in our research to achieve dual-substrate fermentation for erythritol production. The project is divided into two cycles(Liu F etc, 2024; Thuy etc, 2024):

Cycle 1 - Glucose-based erythritol synthesis[Pathway 1]

Cycle 2 - Glycerol-based erythritol synthesis[Pathway 2]

Cycle 1 and Cycle 2 are complementary. Cycle 2 can utilize glycerol as a substrate to produce erythritol only if the proteins, when Cycle 1 are normally expressed. Furthermore, this project provides a solid foundation for future explorations of these techniques in glycerol reuse. The comprehensive and systematic approach outlined herein is critical to navigating the complex regulatory environment.

Design:

Phosphoketolase is a key metabolic enzyme and mainly participates in the sugar metabolism pathways of microorganisms, particularly in the phosphoketolase pathway (Bao,etc.2011). Phosphoketolase is able to catalyzes the scission of phosphoketoses (such as fructose-6-phosphate, etc.) to generate smaller phosphorylated materials, such as glyceraldehyde-3-phosphate (G3P) and acetyl phosphate and acetyl phosplate(Figure 1). Cleavage is associated with the generation of high-energy phosphoryl bonds. For example, acetyl phosphate can act as an energy carrier and directly participate in ATP synthesis (Henard CA, etc. 2015).

Erythritol-4-phosphate dehydrogenase (E4PDH) serves as a key enzyme in the erythritol biosynthetic pathway. It catalyzes the dehydrogenation of erythritol-4-phosphate (E4P), converting it into erythrulose-4-phosphate (E4PuP), accompanied by the concomitant reduction of the cofactors NAD+/NADH or NADP+/NADPH(T. Barbier, etc.2014).

Phosphatase operates (PTase) as an enzyme that removes phosphate groups from its substrates by breaking down phosphate monoesters, which results in phosphate ions and free hydroxyl groups.

In our research, Fructose-6-phosphate is cleaved by phosphoketolase (PK) to produce acetyl-phosphate and erythrose-4-phosphate. Erythrose-4-phosphate is converted to erythritol-4-phosphate by erythritol-4-phosphate dehydrogenase(EPDH). The phosphate group is removed from erythritol-4-phosphate by phosphatase(PTase), yielding the final product erythritol (Figure 1 Pathway 1).

Figure 1.Synthesis pathway of erythritol

(Note: PK: phosphoketolase; EPDH: erythritol-4-phosphate dehydrogenase; PTase: phosphatase; GUT1: Encoding glycerol kinase; GUT2: Encoding glycerol-3-phosphate dehydrogenase; TPI1: Encoding triosephosphate isomerase)

The pCDFDuet vector is a widely used prokaryotic expression system containing two multiple cloning sites (MCS). Each MCS is preceded by a T7lac promoter and a ribosome binding site (RBS), enabling independent induction of target gene expression through IPTG or lactose induction. This vector is particularly suitable for enzyme production in our research project.The plasmid features an N-terminal 6×His tag and a C-terminal S-tag to facilitate subsequent protein purification and verification. Moreover, it carries the CloDF13 replication origin, ensuring stable propagation across various Escherichia coli strains(Figure 2).

Figure 2.The plasmid map

Build:

Currently, we have selected three DNA coding sequences from the National Center for Biotechnology Information (NCBI), namely phosphoketolase (PK), erythritol-4-phosphate dehydrogenase (EPDH), and phosphatase (PTase). These DNA coding sequences were optimized and synthesized according to the codon preference of Escherichia coli. To construct the expression plasmids for PK, EPDH, and PTase, we employed the polymerase chain reaction (PCR) to amplify the coding genes using primers as templates. The obtained PCR products were inserted into the linearized plasmid pCDFDuet.

As shown in Figure 3A, the pCDFDuet-PTase gene was prominently displayed between the 5000 bp and 7500 bp markers, which was in complete agreement with the theoretical length, indicating successful amplification. Figure 3B shows that the EPDH gene was located between the 1000 bp and 1500 bp markers, while the PK gene was between the 1500 bp and 3000 bp markers. The amplified lengths of the genes were consistent with the DNA coding, suggesting successful amplification.

Figure 3A. The electrophoresis verification of the target fragment pCDFDuet-Ptase;Figure 3 B. The electrophoresis verification of the target fragment EPDH and PK; Note: phosphoketolase(PK) -2391bp; Erythritol-4-phosphate dehydrogenas(EPDH)-1509bp; pCDFDuet-phosphatase-5369bp

Next, we cloned three coding DNA (PK, EPDH, and PTase) into the pCDFDuet vector using homologous recombination, and transformed it into E. coli DH5α competent cells. The total length of the three DNA coding is 5565 bp. The electrophoresis result shown in Figure 4A indicates that the amplified product band is located within the range of 5000-7000 bp, which is consistent with the expected fragment size, confirming that the target gene fragment has been successfully amplified. In Figure 4B, the growth of monoclonal colonies with SmR(100ng/ml)was observed, indicating that the transformation experiment was initially effective.By comparing the sequence file with the target gene sequence, the results show that there is no base mutation at the location marked by the red solid arrow, and a base mutation exists at the location marked by the dotted line arrow. The Figure 4C confirm that the target gene fragment has been correctly linked to the vector, further verifying the successful construction of the recombinant plasmid.

Figure 4A. Monoclonal colony verification gel plate; Figure 4B. Monoclonal colony verification petri dish diagram in E. coli DH5α ; Figure 4C. Monoclonal colony verification sequencing plot

Test:

The three-dimensional structures of proteins PK, EPDH and PTase were predicted using AlphaFold2, and the feasibility of the models was evaluated using the metrics ipTM (interface pTM) and pTM (predicted TM-score). The figure 5 shows the three-dimensional structural representation of the protein PK, EPDH and PTase.

Figure 5. Figure A is the three-dimensional structural of the protein PK;Figure B is the three-dimensional structural of the protein EPDH;Figure C is the three-dimensional structural of the protein PTase.

The recombinant plasmids pCDFDuet-PK-EPDH-PTase were transformed into E. coli BL21 (DE3) for expression. A single colony containing the recombinant plasmid was cultured in medium con-taining antibiotics (100 mg/mL SmR ) at 37^oC. When the cul-tures reached optical density (OD600) about 0.6, is o-propy-beta-D-thiogalactopyranoside (0.5mmol and 1mmol IPTG) was added to the broth for induction of the protein in 25^oC . And the recombinant His-6 fusion-type PPE protein was purified by Ni²⁺ affinity chromatography.

As shown in figure 6A, when 0.5 mmol IPTG was used to induce the protein, no PK protein was expressed, but PTase and EPDH were successfully expressed. After being inducted with 1 mmol/L IPTG, both PK, Ptase and EPDH exhibited sig-nificant expression, and the Western blot results showed that both these recombinant prod-ucts could be detected by the anti-His6 antibodies (Figure 6B), suggesting that His6-fused PK, Ptase and EPDH were expressed in E. coli BL21(DE3) successfully.

Figure 6. Optimisation of the culture medium for the soluble expression of the recombinant His6-tagged PK, Ptase and EPDH. (A) SDS-PAGE analysis of the products fermented in media. (B) Western blot analysis of the products fermented in media; Note the Pk protein is 90.6kda. The PTase protein is 59.8kda. The EPDH protein is 56.1kda

Learn:

In this cycle, the plasmids are constructed successfully and transformed in E.coli DH5α. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) experiment and Western Blot(WB) successfully expressed in E.coli BL21(DE3). This cycle experiments proves the success of protein expression before fermentation production, providing essential foundational results for cycle 2.

Design：

Glycerol kinase catalyzes the conversion of glycerol into glycerol-3-phosphate. In adipocytes, due to the absence of glycerol kinase, glycerol generated from lipolysis cannot be utilized directly. Instead, it must be transported via the bloodstream to tissues such as the liver, kidneys, and intestine for further metabolism(Guo L, etc. 2010).

The glyceraldehyde-3-phosphate dehydrogenase (GAPD) is Coenzyme I oxidoreductase with phosphorylation function. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is an NAD-dependent oxidoreductase with phosphorylating activity. It catalyzes the oxidation (dehydrogenation) and phosphorylation of glyceraldehyde-3-phosphate to form 1,3-bisphosphoglycerate. As this reaction represents a central step in sugar metabolism, GAPD plays a critical role in glycolytic pathways(Colell A, etc. 2009).

Triosephosphate isomerase (TPI) is a key enzyme in the glycolytic pathway, catalyzing the reversible isomerization between dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP), and ensuring the efficient progression of glycolysis. (Wang Xiaobin, etc. 2021).

In our research, glycerol is converted to glycerol 3-phosphate by glycerol kinase (GUT1) in Saccharomyces cerevisiae, consuming 1 molecule of ATP. Glycerol 3-phosphate is oxidized to dihydroxyacetone phosphate (DHAP) by glycerol-3-phosphate dehydrogenase (GUT2), with the concomitant reduction of 1 NAD⁺ to NADH. The enzyme triosephosphate isomerase (TPI1) catalyzes the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (G3P). Part of GA-3-P flows into the tricarboxylic acid cycle (TCA cycle) through pyruvate, providing raw materials, energy and cofactors for various life activities of cells. Another part of GA-3-P produced fructose-6-phosphate (F-6-P) under gluconeogenesis and entered the pentose phosphate pathway ( PPP ). The gluconeogenesis pathway includes the intermediates F-6-P and GA-3-P produced by the PPP pathway, which flow to the synthesis of erythritol in pathway 1(Figure 1).

The pETDuet-1 vector harbors the ColE1 replicon, lacI gene and ampicillin resistance gene. This vector can be used in combination with the pCDFDuet vector to achieve co-expression of six target genes in our project within a suitable host strain.The three codeing DNA are inserted into the expression vector pETDuet-1 through homologous recombination (Figure 7).

Figure 7.The plasmid map

Build:

Each target gene was amplified polymerase chain reaction (PCR) using specific primers, and the resulting amplification products were then inserted into the linearized pETDuet vector. The target fragment was ligated to the vector by homologous recombination, transformed into E. coli DH5α, and the expression plasmids were verified.

In Figure 8A, the GUT1 and GUT2 amplification products migrated between the 1500 bp and 3000 bp markers. Similarly, Figure 8B showed that the TPL1 amplification product was located between the 500 bp and 1000 bp markers, and Figure 8C indicates that the pET Duet exhibited a distinct band about 5000 bp. The observed lengths of all amplified genes were consistent with their respective coding sequences, validating the success of the amplification process.

Figure 8A. The electrophoresis verification of the target fragment pET Duet(5462bp). Figure 8B. The electrophoresis verification of the target fragments GUT1(2130bp)and GUT2(1950bp).Figure 8C. The electrophoresis verification of the target fragment TPL1(747bp) .

The total length of the coding GUT1, GUT2, and TPL1 is 4827bp. As shown in the electrophoresis result in figure 9A, the amplified product presents a clear band at approximately 5000 bp, which is highly consistent with the expected fragment size. The growth of monoclonal colonies figure 9B with Amp(100ng/ml) was observed. And Figure 9C confirmed that the target gene fragment has been correctly linked to the vector, further verifying the success of the construction of the recombinant plasmid.

Figure 9A. Monoclonal Colony Verification Gel Plate; Figure 9 B. Monoclonal Colony Verification Petri Dish Diagram in E. coli DH5α; Figure 9C. Monoclonal colony verification sequencing plot

Test:

The three-dimensional structures of proteins GUT1,GUT2 and TPl1 were predicted using SWISS-MODEL, and the feasibility of the models was evaluated using the metrics GMQE (Global Model Quality Estimation) and QMEAN. The figure10 shows the three-dimensional structural representation of the protein GUT1,GUT2 and TPl1.

Figure 10. Figure A is the three-dimensional structural of the protein GUT1;Figure B is the three-dimensional structural of the protein GUT2;Figure C is the three-dimensional structural of the protein TPL1.

The recombinant plasmids pET Duet-GUT1-GUT2-TPl1 were transformed into E. coli BL21 (DE3) for expression. A single colony containing the recombinant plasmid was cultured in medium con-taining antibiotics (100 mg/mL Amp) at 37^oC. The other protein expression conditions are consistent with pCDFDuet-PK-EPDH-PTase. The figure 11A shown that the TPL1,GUT2 and GUT1 exhibited sig-nificant expression after being inducted with 0.5 and 1 mmol/L IPTG. And the Western blot results showed that both these recombinant prod-ucts could be detected by the anti-His6 antibodies (Figure 11B), suggesting that His6-fused TPL1,GUT2 and GUT1 were expressed in E. coli BL21(DE3) successfully.

Figure 11. Optimisation of the culture medium for the soluble expression of the recombinant His6-tagged TPL1,GUT2 and GUT1. (A) SDS-PAGE analysis of the products fermented in media. (B) Western blot analysis of the products fermented in media; NoteThe size of the GUT1 protein is 79.8kda. The size of the GUT2 protein is 72.7kda. The size of the TPI1 protein is 26.8kda

1. Plasmid co-transfection

The plasmid pCDFDuet-PK-EPDH-PTase are abbreviated as PPE; The plasmids pET Duet-GUT1-GUT2-TPl1 are abbreviated as GGT. The plasmids PPE and GGT were transfed to Escherichia coli BL21. Monoclonal colonies were screened using 100 mg/ml Ampicillin (Amp⁺) and Streptomycin Resistance (SmR) antibiotics. In figure 12, single colonies have grown.

Figure 12. Monoclonal colony culture dish diagram in Escherichia coli BL21

As shown in figure 13, lanes 1 to 8 correspond to the amplification products of PPE1, GGT1, PPE2, GGT2, PPE3, GGT3, PPE4, and GGT4. The results indicate that the amplification bands of PPE4 and GGT4 are present within the molecular weight range of 2000 bp to 4000 bp. Moreover, their actual sizes are exactly consistent with the theoretically expected lengths, thereby confirming that these two target fragments have been successfully amplified.

Figure 13. Monoclonal colony verification. note:The length of GGT is 3726 bp, and the length of PPE is 3504 bp

2. Growth Curve

The plasmids PPE and GGT were transfed to Escherichia coli BL21. The OD600 values were measured three times at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, 20 hours, 24 hours, 30 hours, and 48 hours, respectively. As can be seen from figure 14, from 0 to 12 hours, the OD600 values of both CK(Blank control) and co-transformation strains increased linearly. From 12 hours to 48 hours, the OD600 values gradually stabilized. In figure 14, the CK groups were not significant difference to compared with bacterial strain pCDFDuet-PK-EPDH-PTase and pET Duet-GUT1-GUT2-TPl1 . The coding genes PK, EPDH, PTase, GUT1, GUT2, and TPl1 don’t affect the growth of the strain.

Figure 14. The growth curve of pCDF Duet-PK-EPDH-PTase+ pET Duet-GUT1-GUT2-TPl1 and CK. Note:The plasmid pCDFDuet-PK-EPDH-PTase are abbreviated as PPE; The plasmids pET Duet-GUT1-GUT2-TPl1 are abbreviated as GGT

3. Determination of erythritol production by HPLC

We prepared five different fermentation media with varying substrate compositions and ratios (fermentation conditions: 37°C, pH 7-8), as follows: Glucose:Glycerol = 10:10 (g/L), Glucose:Glycerol = 20:10 (g/L), Glucose:Glycerol = 10:20 (g/L), Glucose:Glycerol = 20:0 (g/L), Glucose:Glycerol = 0:20 (g/L). Samples were taken at different time points (5h, 16h, 24h, 48h) to measure the concentrations of glucose, glycerol, and erythritol.The concentrations of products and substrates were determined by high-performance liquid chromatography (HPLC). After sampling, centrifugation was performed to collect the supernatant (at 8000 r/min for 5 minutes), and it was filtered through a 0.22 μm membrane. The Aminex HPX-87H analytical column (300 mm ×7.8 mm) (from Bio-Rad, USA), 2414 differential refractive index detector (from Waters, USA). The detection conditions were column temperature 35 °C, injection volume 10 μL, mobile phase 5 mmol/L sulfuric acid, flow rate 0.6 mL/min. All the diagrams were completed using GraphPad.

According to figure 15A, the glucose concentration gradually declines with time in Glucose: Glycerol = 20:0 g/L. The concentration of erythritol increased over time, while no erythritol was produced in the control group(CK-E.coli BL21). This indicates that genes PK, EPDH, and PTase were successfully expressed, and the engineered strain demonstrated the capability to utilize glucose as a carbon substrate for fermentative production of erythritol.

In figure 15B, The concentration of glycerol gradually decreased, while the concentration of erythritol showed an upward trend over time in the Glucose: Glycerol = 0:20 g/L. However, no erythritol was produced in the control group. This indicates that the genes PK, EPDH, PTase, GUT1, GUT2, and TPI1 were successfully co-expressed in the engineered strain, and the glycerol and glucose metabolic pathways were properly established. Furthermore, it demonstrates that the strain can utilize a complete glycerol-based medium for fermentative erythritol production.

Figure 15. The concentration of erythritol, glucose and glycerol (A). The relationship graph of glucose, glycerol, and erythritol with time in Glucose: Glycerol = 20:0 g/L. (B) the relationship graph of glucose, glycerol, and erythritol with time in the Glucose: Glycerol = 0:20 g/L.

The figure 16A and B demonstrate that no erythritol was produced in the CK group (blank control), while the glucose concentration showed a decreasing trend over time in both the Glucose:Glycerol = 20:10 g/L and Glucose:Glycerol = 10:20 g/L. The glycerol concentration exhibited no significant change from 5h to 24 h, followed by a decrease between 24 and 48 hours. At Glucose:Glycerol = 20:10 g/L and Glucose:Glycerol = 10:20 g/L, erythritol concentration accumulation increased with time.

These results indicate that the strain likely preferentially utilizes glucose as the primary substrate during the 5-24 hour period at glucose and glycerol ratios of 2:1 and 1:2. When the glucose concentration becomes sufficiently low, the strain further metabolizes glycerol as a secondary substrate for erythritol production. This confirms that our engineered strain can utilize dual substrates (glucose and glycerol) as carbon sources for fermentative erythritol biosynthesis.

Figure 16. The concentration of erythritol, glucose and glycerol. (A). The relationship graph of glucose, glycerol, and erythritol with time in Glucose: Glycerol = 20:10 g/L. (B) the relationship graph of glucose, glycerol, and erythritol with time in the Glucose: Glycerol = 10:20 g/L.

According to figure 17, the concentration of glucose gradually decreases over time in Glucose: Glycerol = 10:10 g/L, but the concentration of glycerol shows no significant trend from 5 to 24 hours, and then decreases from 24 to 48 hours in Glucose: Glycerol = 10:10 g/L. The concentration of erythritol increases over time. These results indicate that glucose is preferentially utilized as the carbon source at glucose and glycerol ratios of 1:1, 1:2 and 2:1, and erythritol production occurs under all these conditions.

Figure 17. The concentration of erythritol, glucose and glycerol in Glucose: Glycerol = 10:10 g/L.

To enhance erythritol production and investigate the effects of varying glucose-to-glycerol ratios in the culture medium on erythritol yield, we utilized MATLAB software to perform 3D modeling predictions based on existing data. This model predicts erythritol production levels after fermentation using different proportional combinations of glucose and glycerol as carbon sources.A three-dimensional model was constructed by performing surface fitting on each discrete data point, and based on this model, the prediction analysis of the highest and lowest points was realized.

The Y-axis is labeled as "Time (h)" in the figure 18. The X-axis is labeled as "Glucose: Glycerol", displaying different ratio values such as 0:20, 10:10, 20:0, etc. The Z-axis is labeled as "Erythritol (g/L)". The figure 17 shown that the highest value is (1.7676, 39.7474, 3.403643), and the lowest value is (0; 0; 0). At the highest value, the ratio of glucose concentration to glycerol concentration was 17:10. After the initial measurement was completed, the samples were subjected to a secondary fermentation process, and then the highest values were measured using high-performance liquid chromatography (HPLC) technology.

Figure 18. 3D modeling of the production volume of erythritol

In figure 19, the concentration of glucose gradually decreases over time, but the concentration of glycerol shows no significant trend from 5h to 24h, and then decreases from 24 to 48 hours in Glucose: Glycerol = 10:10 g/L. The concentration of erythritol increases over time. These results indicate that glucose is preferentially utilized as the carbon source at glucose and glycerol ratios of 1:1, 1:2, 2:1and 1.7:1, and erythritol production occurs under all these conditions.

Figure 19. the relationship graph of glucose, glycerol, and erythritol with time atGlucose: Glycerol = 17:10 g/L

After 48 hours of fermentation in media with varying ratios of glucose to glycerol, the consumption of glucose and glycerol, as well as the erythritol production yield and conversion rate, were calculated based on data determined by HPLC. After 48 hours, the strain consumed 13.13g glucose , and produced 2.33g erythritol with a conversion rate of 17.74% at Glucose: Glycerol = 20:0 g/L. At Glucose: Glycerol = 20:10 g/L, the strain consumed18.73g glucose and5.03g of glycerol, and produced 2.82g erythritol with a conversion rate of 11.85% . The experimental data for other groups are presented in Table 1. The table 1 shows that the experimental group with the highest erythritol yield was Glucose:Glycerol = 17:10 g/L, followed by Glucose:Glycerol = 10:20 g/L. The group with the lowest erythritol yield was Glucose:Glycerol = 20:0 g/L. In Table 1, the highest erythritol conversion rate was 20.99% in the Glucose:Glycerol = 0:20 g/L, while the lowest was 11.85% in the Glucose:Glycerol = 20:10 g/L .

Table 1. Under different ratios of glucose and glycerol, the concentrations and conversion rates of glucose, glycerol, and erythritol

Learn:

We have developed a dual-substrate production system by employing molecular biological techniques to enhance and integrate the glucose and glycerol metabolic pathways within the bacterial. This innovative approach offers a novel production paradigm characterized by environmental sustainability, operational safety, and reduced by product formation, thereby enabling efficient utilization of glycerol resources.

The experimental results demonstrated that the engineered strain can utilize both glucose and glycerol as mixed substrates for erythritol production. We further investigated the effects of varying glucose-to-glycerol ratios on erythritol yield, which revealed the strain's preferential utilization of glucose over glycerol. However, the optimal substrate ratio for maximizing production yield was not identified in this study.Overall, our study provides both technical and data support for the industrial production of erythritol.

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